Mycobacterium tuberculosis is a pathogen that is able to adapt to a variety of different environments encountered during the progressive course of human infection. An ability to inhibit maturation of the macrophage phagosome initially created an ideal environment for bacterial growth, allowing colonization of the body. when deprived of this favorable environment by the hosts's own tissue-damaging immune response, infection often fails to progress, and bacilli enter an nonreplicating latent state, having reached a degree of equilibrium with the host. Changes in the host over time may then allow bacteria to resume replication, leading to further tissue destruction and extracellular growth to high titers. This project will identify bacterial genes that are specifically expressed by M. tuberculosis during adaptation to these in vivo environments. A new method developed specifically for examination of mycobacterial mRNAs expressed in infected host cells and tissues (SCOTS) has so far identified 9 M. tuberculosis genes which are expressed in response to growth within cultured human macrophage phagosomes. The first aim of the proposed work is to make bacterial strains specifically inhibited in expression of these genes and evaluate their ability to survive and grow in cultured human macrophages. The second aim is to identify additional M. tuberculosis genes that are differently expressed by tubercle bacilli in another environment that bacilli normally encounter during the natural course of human infection. Bacterial genomic array hybridization with cDNAs obtained by SCOTS will be used to analyze global mRNA expression patterns in bacilli recovered from patient sputum samples, providing insight into the physiology and metabolism of the microbe during active growth in the human lung. Our third aim is to extend analysis of differential bacterial gene expression to a C57BL/6 mouse model of host interaction, facilitating studies of both active and latent types of infection in a genetically defined host. Bacterial cDNA already obtained by SCOTS from tubercle bacilli growing in cultured mouse macrophages and from infected mouse lung tissues will be compared by array hybridization to cDNA from tubercle bacilla growing in human macrophages and lung tissues. A limited number of M. tuberculosis genes commonly expressed in response to these mammalian host interactions will then be evaluated for their contributions to virulence in this animal model. Understanding the roles of such differentially expressed genes will further define host-pathogen interactions in human disease, and allow development of new tools to reduce the enormous global impact of tuberculosis on mankind.